The present invention relates to transgenic plants exhibiting increased tolerance to stress and methods of generating same.
Modern agriculture strives to achieve the highest possible crop yields in order to overcome the continuously growing land limitation. Uniformity, as well as growth density, render modern crops susceptible to quickly spreading damage of many pathogens such as nematodes, bacteria, fungi, viruses, viroids, and phytoplasms. A growing resistance exists to the use of chemical pesticides due to many disadvantages brought forth by chemical abuse including negative environmental effects, and diminishing affectivity. For example, the magnitude of fungicidal treatments has provoked the appearance of resistant strains, necessitating the development of new treatments (Leroux et al., Pest Manag. Sci. 58:876, 2002). On the other hand, fighting pathogens by utilizing the biological inert, plant mechanisms is environmentally safer, and less prone to become ineffective by the creation of resistant pathogenes.
An example of a damaging plant pathogen is B. cinerea. This phytopathogenic fungi has a broad host range, of more than 200 plant species, including tomato (Elad et al., In: Botrytis: Biology, Pathology and Control, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 1-8, 2004). B. cinerea cause rapid destruction of the host plant tissues as it proceeds to colonize it (a pathology called necrotrophy). Together with other filamentous fungi, it is considered to be the principal pathogenic agents of plants. Estimated losses for vineyards in France amount to 15-40% of the harvest, depending on climatic conditions. Other losses are estimated at 20-25% of the strawberry crops in Spain and cut flowers in Holland. Fungicidal treatments against B. cinerea cost about 540 million euros in 2001, which represents 10% of the world fungicide market (Annual Report, UIPP, 2002). Other plant pathogens are viruses, e.g., the tobacco mosaic virus (TMV), the potato virus Y (PVY) and tomato yellow leaf curl virus (TYLCV). Plant virus diseases pose severe constraints to the production of a wide range of economically important crops worldwide (Agrios, G N, Plant Pathology. fourth ed. Academic Press, Inc., San Diego, Calif., 1997). Some estimates put total worldwide damage due to plant viruses as high as 6×1010 US$ per year. Diseases caused by plant viruses are difficult to manage and their control mainly involves the use of insecticides to kill insect vectors, the use of virus-free propagating materials, and the selection of plants with appropriate resistance genes. Virus-free stocks are obtained by virus elimination through heat therapy and/or meristem tissue culture, but this approach is ineffective for viral diseases transmitted by vectors. While vectors can be controlled by insecticides, often the virus has already been transmitted to the plant before the insect vector is killed. The use of resistant cultivars has been the most effective means of control, however plant virus resistance genes are frequently unavailable and their introgression into some crops is not straightforward.
Abiotic stress (also referred to as “environmental stress”) conditions such as salinity, drought, flood, suboptimal temperature and toxic chemical pollution, are additional major factors which cause substantial damage to agricultural plants. Most plants have evolved strategies to protect themselves against these conditions. However, most of the crop plants are very susceptible to abiotic stress, and thus necessitate optimal growth conditions for commercial crop yields. Furthermore, crop plants are in numerous times grown outside of the climate from which they originate. The unnatural conditions, together with the sensitivity of crop plants, effect plant development and growth which result in a less then optimum yield. An example of abiotic stress is excessive heat, which, in most times, is linked to drought. Germination of many crops is very sensitive to temperature. Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function (Buchanan et al., in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md., 2000). Heat shock may produce a decrease in overall protein synthesis, accompanied by expression of heat shock proteins.
Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are part of protein-lipid structures residing in fibrillar-type chromoplasts, such as those of flowers and ripening fruit, as well as in other plastids. For example, ChrC, a 35-kD carotenoid-associated PAP, was found to be expressed in chromoplasts of fruit and flower tissues of the yellow cucumber, Cucumis sativus (Vainstein et al., Plant Physiol. 104, 321-326, 1994; Vishnevetsky et al., Plant J. 10, 1111-1118, 1996). PAPs, like the Cucumber ChrC and, the pepper PAP Fib, are known to accumulate at both protein and transcript levels, in parallel to carotenoid pigment accumulation, as part of the differentiation of chloroplasts to non photosynthetic chromoplasts (chromoplastogenesis), and in concomitance with fibril development (Deruere et al., Plant Cell 6, 119-133, 1994). Interestingly, PAPs, like the potato CDSP34 and pepper fibrillin, were found to be overexpressed upon induction of abiotic stresses e.g., oxidative stress, light, salt, wound, aging and drought [examples can be found in Chen et al., Plant J. 14, 317-326, 1998; Langenkämpel et al., J Exp Bot 52(360): 1545-1554 (2001); Murphy D J, Proceedings of the 16th International Plant Lipid Symposium, Budapest, pp. 55-62, 2004]. Elevation of expression of PAP upon stress induction was also evidenced in experiments showing higher expression of exogenous promoters in transgenic plants; for example, the expression of the fibrillin promoter in transgenic tomato plants was elevated during bacterial (Erwinia strains) infections [Langenkämpel et al., J Exp Bot 52(360): 1545-1554 (2001)] and during the induction of abiotic stresses e.g., drought, cold, salt, light and herbicides (Manac'h and Kuntz, Plant Physiol. Biochem. 37, 859-868, 1999). Hence, it is suggested that PAP expression is increased upon abiotic stress but no direct evidence is provided showing that PAP may confer resistance and is not, a mere “by product” of stress induction.
Indeed, up to date, overexpression of Fib in transgenic tobacco, and fibrillin in fibrillin overexpressing Arabidopsis lines, was merely found to improve plant performance under induced light stress conditions (Rey et al., Plant J. 21, 483-494 2000; Yang et al., PNAS 103: 6061-6066, 2006). No other support was provided to date regarding the ability of PAP to confer tolerance to other abiotic stress, needless to say to biotic stress.
The cucumber ChrC promoter was characterized and used to develop products for increasing accumulation and sequestration of carotenoids in plants and bacteria (U.S. Pat. No. 6,551,793). Two factors were found to activate the ChrC promoter. The first, GA3, which plays a critical role in chromoplastogenesis, was found to lead to enhanced carotenoid accumulation as well as to transcriptional activation of ChrC expression. The response to GA was localized to a 290-bp fragment within the ChrC promoter (Vishnevetsky et al., Plant J. 20, 423-431, 1999; Sutoh K and Yamauchi D, Plant J. 34, 635-645, 2003). Another activator of the ChrC promoter, a myb-like factor termed MYBYS, was recently characterized, and harnessed to develop a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants (Tzfira et al., Plant Mol. Biol. 57, 503-516, 2005).
Use of the ChrC promoter was also suggested for inducing flower-specific expressed genes in the genus Targets (U.S. Patent Application 0060162020).
In none of the abovementioned studies, however, was the use of the coding sequence of PAPs suggested for improving plant pathogen resistance, nor was the use of the PAP or PAP expression activators, suggested for conferring abiotic stress resistance excluding light resistance.
There is thus a widely recognized need for, and it would be highly advantageous to have, constructs for conferring resistance to stress.